Competition binding assay for detecting P2YADP receptor ligands

ABSTRACT

The invention provides a competition binding assay for detecting P2Y ADP  receptor ligands.

It is known that ADP (adenosine-5′-diphosphate) plays a pivotal role interms of platelet function by causing adhesion, degranulation, shapechange and aggregation of platelets via interaction with cell surface P2receptors. It is the P2Y_(ADP) receptor (formerly known as P_(2T)) thatis primarily involved in mediating platelet aggregation, which is an asyet uncloned G-protein linked receptor. The pharmacologicalcharacteristics of this receptor have been described, for example, inthe references by Humphries et al., Br. J. Pharmacology, (1994), 113,1057-1063, and Fagura et al., Br. J. Pharmacology, (1998), 124, 157-164.

Compounds having antagonist activity at the P2Y_(ADP) receptor areknown, for example, from WO 98/28300, WO 97/03084, WO 94/18216 andEP-A-508 687 and are useful as anti-thrombotic agents in the treatmentor prophylaxis of diseases such as unstable angina, coronary angioplasty(PTCA) and myocardial infarction.

It would be desirable to identify further compounds with bindingactivity at the P2Y_(ADP) receptor, and also to identify furthertissues/cell lines containing this receptor.

In accordance with the present invention, there is therefore provided acompetition binding assay which comprises contacting a P2Y_(ADP)receptor, preferably a human P2Y_(ADP) receptor, with a P2Y_(ADP)receptor radioligand and a candidate P2Y_(ADP) receptor ligand, andmeasuring bound radioactivity.

The assay according to the present invention is very convenientlycarried out on multi-well microtitre plates, thereby enabling a fast,simple and reproducible way of screening large numbers of potentialP2Y_(ADP) receptor ligands.

In the context of the present specification, the term “P2Y_(ADP)receptor ligand”, unless otherwise indicated, defines a ligand, e.g. anagonist or antagonist, of the P2Y_(ADP) receptor other than anaturally-occurring ligand. The ligand may, for example, be a chemicalcompound, or a salt or solvate thereof.

The radioligand used in the assay is a substance which binds to theP2Y_(ADP) receptor and which may be synthesised containing one or moreradioactive atoms. Examples of substances which when radiolabelled maybe used as the radioligand include:

(a) Compounds of formula (I)

 wherein

X is OH or NHR³;

R¹ is C₁₋₆-alkyl, C₃₋₈-cycloalkyl or a phenyl group, each group beingoptionally substituted by one or more halogen atoms and/or OR⁴, NR⁴R⁵,C₁₋₆-thioalkyl and/or C₁₋₆-alkyl (itself optionally substituted by oneor more halogen atoms); R² is C₁₋₈-alkyl or C₂₋₈-alkenyl each of whichis optionally substituted by one or more halogen atoms and/or OR⁴,NR⁴R⁵, C₁₋₆-thioalkyl, C₃₋₈-cycloalkyl, aryl and/or C₁₋₆-alkyl groups;or R² is a C₃₋₈-cycloalkyl group optionally substituted by one or morehalogen atoms and/or OR⁴, NR⁴R⁵, C₁₋₆-thioalkyl, phenyl and/orC₁₋₆-alkyl groups; the optional phenyl substituent being furtheroptionally substituted by one or more halogen atoms and/or NO₂, C(O)R⁴,OR⁴, NR⁴R⁵, C₁₋₆-thioalkyl and/or C₁₋₆-alkyl groups;

R³ is hydrogen or C₁₋₆-alkyl substituted by one or more hydroxy and/orphenyl groups and optionally by one or more halogen atoms, wherein thephenyl group is substituted by one or more hydroxy groups and optionallysubstituted by one or more halogen atoms and/or NO₂, C(O)R⁴, OR⁴, NR⁴R⁵,C₁₋₆-thioalkyl and/or C₁₋₆-alkyl groups, or R³ is a C₁₋₆-alkyl groupsubstituted by a C(O)NR⁴R⁵ or a COOH group and optionally by one or morehalogen atoms and/or OR⁴, C(NH)NR⁴R⁵, C(O)NR⁴R⁵, phenyl and/orC₁₋₆-alkyl groups, wherein the alkyl group is optionally substituted byone or more hydroxy and/or phenyl groups and wherein the phenyl group isoptionally substituted as defined above for R³; or

R³ is a lactam ring of formula (i):

 wherein Q is a (CH₂)_(m) moiety wherein m is 1, 2 or 3, Z is O, C(O) orCH₂;

R⁴ and R⁵ each independently represent hydrogen, phenyl or a C₁₋₆-alkylwherein the alkyl group is optionally substituted by one or more phenylgroups; and salts and solvates thereof;

(b) Compounds of formula (II)

 wherein

B is O or CH₂;

X is selected from NR¹R², SR¹ and C₁-C₇ alkyl;

Y is selected from NR¹R², SR¹ and C₁-C₇ alkyl;

R¹ and R² is each and independently selected from H, or C₁-C₇ alkyloptionally substituted upon or within the alkyl chain by one or more ofO, S, N or halogen;

R³ and R⁴ are both hydrogen, or R³ and R⁴ together form a bond;

A is COOH, C(O)NH(CH₂)_(p)COOH, C(O)N[(CH₂)_(q)COOH]₂,C(O)NHCH(COOH)(CH₂)_(r)COOH or 5-tetrazolyl, wherein p, q and r is eachand independently 1, 2 or 3; and salts and solvates thereof;

(c) Compounds of formula (I)

 wherein

R¹ and R² independently represent hydrogen or halogen;

R³ and R⁴ independently represent phenyl, or C₁-C₆ alkyl optionallysubstituted by one or more substituents selected from OR⁵, C₁-C₆alkylthio, NR⁶R⁷, phenyl, COOR⁸ and halogen;

R⁵, R⁶, R⁷ and R⁸ independently represent hydrogen or C₁-C₆ alkyl;

X represents an acidic moiety; and salts and solvates thereof;

(d) Compounds of formula (IV)

 wherein

Q represents CR¹R²;

R represents O or CR³R⁴;

W represents O or CH₂;

R¹, R², R³ and R⁴ independently represent hydrogen or halogen;

X represents S(O)_(n)R⁵, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ acylamino,CONR⁶R⁷, NR⁸R⁹, halogen, a 5- or 6-membered S containing heterocycle, orphenyl optionally substituted by C₁-C₆ alkyl;

n represents 0, 1 or 2;

R⁵ represents aryl or C₁-C₆ alkyl optionally substituted by one or moresubstituents selected from hydroxy, C₁-C₆ alkoxy, halogen and aryl;

R^(6, R) ⁷, R⁸ and R⁹ independently represent hydrogen or C₁-C₆ alkyl;

Y represents NH₂ or C₁-C₆ alkoxy;

Z represents an acidic moiety;

in addition, when R represents CR³R⁴, then —Q—Z may also representhydroxy or —OP(O)(OH)₂, provided that:

i) when R is O, W is O, X is Cl, Y is NH₂ and Z is —P(O)(OH)₂, thenCR¹R² does not represent CH₂; and

ii) when R is O, W is O, X is SCH₃, Y is NH₂ and Z is —P(O)(OH)₂, then—CR¹R² does not represent CH₂, CF₂ or CCl₂; and salts and solvatesthereof;

(e) Compounds of formula (V)

 wherein

R¹ is a C₁₋₆ alkyl, C₃₋₈-cycloalkyl or a phenyl group, each group beingoptionally substituted by one or more substituents selected fromhalogen, OR⁸, NR⁹R¹⁰, SR¹¹ or C₁₋₆ alkyl (itself optionally substitutedby one or more halogen atoms);

R² is C₁₋₈ alkyl optionally substituted by one or more substituentsselected from halogen, OR⁸, NR⁹R¹⁰, SR¹¹, C₃₋₈-cycloalkyl, aryl(optionally substituted by one or more alkyl groups and/or halogenatoms), or C₁₋₆-alkyl; or R² is a C₃₋₈-cycloalkyl group optionallysubstituted by one or more substituents selected from halogen, OR⁸,NR⁹R¹⁰, SR¹¹, C₁₋₆-alkyl or phenyl (the latter two being optionallysubstituted by one or more substituents selected from halogen, NO₂,C(O)R⁸, OR⁸, SR¹¹, NR¹²R¹³, phenyl and C₁₋₆-alkyl which is optionallysubstituted by one or more halogen atoms);

one of R³ or R⁴ is hydroxy and the other is hydrogen, hydroxy or NR⁹R¹⁰;

R⁵ is (CH₂)_(n)NR¹⁴R¹⁵ where n is 0 to 6 and R¹⁴ and R¹⁵ areindependently hydrogen, C₁₋₆-alkyl or phenyl; or R⁵ is CONR¹⁶R¹⁷ whereR¹⁶ is hydrogen or C₁₋₆-alkyl, and R¹⁷ is C₁₋₆-alkyl or C₃₋₆-cycloalkyleach of which is substituted by NR¹⁸R¹⁹ and optionally substituted byphenyl, or R¹⁷ is C₁₋₆-alkyl or C₃₋₆-cycloalkyl substituted by phenylwhich is substituted by NR¹⁸R¹⁹ where R¹⁸ and R¹⁹ are independentlyhydrogen, C₁₋₆-alkyl or phenyl; or R¹⁷ is a 5- to 8-membered saturatedheterocycle containing one or more nitrogen atoms and optionallysubstituted on nitrogen by hydrogen, C₁₋₆-alkyl or phenyl;

or R¹⁶ and R¹⁷ together with the nitrogen atom to which they areattached form a 5- to 8-membered ring which is substituted by NR¹⁸R¹⁹ asdefined above; or

R¹⁶ together with R¹⁹ forms a 6- to 8-membered ring containing the twonitrogen atoms in which R¹⁷ and R¹⁸ are as defined above; or R⁵ is(CH₂)_(p)NR²⁰CO(CH₂)_(q)OR²¹ or (CH₂)_(p)NR²²(CH₂)_(q)NR²³COR²⁴ where pand q are independently 1 to 4 and R²⁰, R²¹, R²² R²³ and R²⁴ areindependently C₁₋₄-alkyl or phenyl; or R⁵ is CH═CHCH₂NR²⁵R²⁶ where R²⁵is hydrogen, C₁₋₆ alkyl or phenyl and R²⁶ is hydrogen or(CH₂)_(y)NR²⁷R²⁸ where y is 2-4 and R²⁷ and R²⁸ are independentlyhydrogen, C₁₋₆ alkyl or phenyl;

R⁸, R⁹, R¹⁰ and R¹¹ are independently hydrogen or C₁₋₆-alkyl; and

R¹² and R¹³ are independently hydrogen, C₁₋₆-alkyl or acyl groups;

and salts and solvates thereof;

(f) Compounds of formula (VI)

 wherein

R¹ is a C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₈-cycloalkyl or aphenyl group, each group being optionally substituted by one or moresubstituents selected from halogen, OR⁸ NR⁹R¹⁰, SR¹¹ or C₁₋₆ alkyl(itself optionally substituted by one or more halogen atoms); R² is C₁₋₈alkyl optionally substituted by one or more substituents selected fromhalogen OR⁸, NR⁹R¹⁰, SR¹¹, C₃₋₈-cycloalkyl, aryl (optionally substitutedby one or more alkyl groups and/or halogen atoms), or C₁₋₆-alkyl; or R²is a C₃₋₈-cycloalkyl group optionally substituted by one or moresubstituents selected from halogen, OR⁸, NR⁹R¹⁰, SR¹¹, C₁₋₆-alkyl orphenyl, the latter two groups being optionally substituted by one ormore substituents selected from halogen, NO₂, C(O)R⁸, OR⁸, SR¹¹,NR¹²R¹³, a fused 5- or 6-membered saturated ring containing one or twooxygen atoms, phenyl or C₁₋₆-alkyl the latter two groups beingoptionally substituted by OR⁸, NR⁹R¹⁰ or one or more halogen atoms;

one of R³ and R⁴ is hydroxy and the other is hydrogen, hydroxy orNR⁹R¹⁰;

R is a group (CR⁵R⁶)_(m)OR⁷ where m is 0 or 1, R⁵ and R⁶ areindependently hydrogen, C₁₋₆ alkyl or phenyl the latter two groups beingoptionally substituted by halogen, and R⁷ is hydrogen, C₁₋₆ alkyl or(CR⁵R⁶)R¹⁴ where R⁵ and R⁶ are as defined above, n is 1 to 3 and, R¹⁴ isCOOH, OR¹⁵, NR¹⁵R¹⁷ or CONR¹⁶R¹⁷;

or R is a C₁₋₄ alkyl or C₂₋₄ alkenyl group, each of which is substitutedby one or more groups selected from ═S, ═O, ═NR²⁰ or OR²¹ and optionallysubstituted by one or more groups selected from halogen, C₁₋₄ alkyl,phenyl, SR²¹, NO₂ or NR²²R²³ (where R²¹, R²², and R²³ are independentlyhydrogen, C₁₋₄ alkyl or phenyl; R²⁰ is OR²⁴ or NR²⁵R²⁶, where R²⁴ ishydrogen, C₁₋₄ alkyl or phenyl, and R²⁵ and R²⁶ are independentlyhydrogen, C₁₋₄ alkyl, aryl, C₁₋₆ acyl, arylsulphonyl or arylcarbonyl);

R⁸ is hydrogen, C₁₋₆ alkyl optionally substituted by halogen or R⁸ isphenyl optionally substituted by one or more substituents selected fromhalogen, NO₂, C(O)R⁶, OR⁶, SR⁹, NR¹⁰OR¹¹;

R⁹, R¹⁰ and R¹¹ are independently hydrogen or C₁₋₆ alkyl;

R¹² and R¹³ are independently hydrogen, C₁₋₆ alkyl, acyl, alkyl sulfonyloptionally substituted by halogen, or phenyl sulfonyl optionallysubstituted by C₁-C₄ alkyl; and R¹⁵, R¹⁶ and R¹⁷ are independentlyhydrogen or C₁₋₆ alkyl; and salts and solvates thereof; and

(g) Compounds of formula (VII)

 wherein

R¹ is a C₁₋₆ alkyl, C₃₋₈-cycloalkyl or a phenyl group, each group beingoptionally substituted by one or more substituents selected fromhalogen, OR⁶, NR⁷R⁸, SR⁹ or C₁₋₆ alkyl (itself optionally substituted byone or more halogen atoms);

R² is C₁₋₈ alkyl optionally substituted by one or more substituentsselected from halogen, OR⁶, NR⁷R⁸, SR⁹, C₃₋₈-cycloalkyl, aryl(optionally substituted by one or more alkyl groups and/or halogenatoms), or C₁₋₆-alkyl; or R² is a C₃₋₈-cycloalkyl group optionallysubstituted by one or more substituents selected from halogen, OR⁶,NR⁷R⁸, SR⁹, C₁₋₆-alkyl or phenyl (the latter two being optionallysubstituted by one or more substituents selected from halogen, NO₂,C(O)R⁶, OR⁶, SR⁹, NR¹⁰R¹¹, phenyl and C₁₋₆-alkyl which is optionallysubstituted by one or more halogen atoms);

one of R³ or R⁴ is hydrogen and the other is hydroxy;

X is OH or NHR⁵;

R is a C₁₋₆-alkyl group substituted by COOH or C(O)NR⁷R⁸ and optionallyby one or more further substituents selected from halogen, OR¹²,C(NH)NR¹³R¹⁴, C(O)NR¹⁵R¹⁶, phenyl (optionally substituted by one or moregroups selected from halogen, NO₂, C(O)R⁶, OR²⁰, NR⁷R⁸, SR⁹ andC₁₋₆-alkyl) or C₁₋₆-alkyl (optionally substituted by one or more hydroxyor phenyl groups);

or R⁵ is a lactam ring of formula (i):

 wherein Q is a (CH₂)_(m) moiety where m is 1, 2 or 3, Z is O, C(O) orCH₂ and R¹⁸ is hydrogen or C₁₋₆-alkyl;

R⁶, R⁹, R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently hydrogen orC₁₋₆-alkyl;

R⁷ and R⁸ are independently hydrogen, C₁₋₆-alkyl (optionally substitutedby one or more phenyl groups) or phenyl groups; and

R¹⁰ and R¹¹ are independently hydrogen, C₁₋₆-alkyl or acyl groups;

and salts and solvates thereof.

Compounds of formulae (I), (II), (III), (IV), (V), (VI) and (VII) aredisclosed respectively in WO 98128300, WO 97/03084, WO 94/18216,EP-A-508 687, PCT/SE98/01392, PCT/SE98/01393 and PCT/SE98/01394 and thecontents of these seven documents are incorporated herein by reference.

Examples of suitable salts that may be used include alkali metal (e.g.sodium or potassium), alkaline earth metal (e.g. calcium or magnesium),Group III metal (e.g. aluminium), ammonium, hydrochloride, hydrobromide,phosphate, acetate, fumarate, maleate, tartrate, citrate, oxalate,methanesulphonate and p-toluenesulphonate salts.

The techniques for radiolabelling substances may be those conventionallyused in the art and therefore the radioligand may be prepared by methodsknown in the art.

The radioligand is most preferably a radiolabelled compound of formula(I) or(II) or a salt or solvate thereof, and is especially[¹²⁵I]-[1S-[1α,2β,3β,4α(E)]]-2,3-dihydroxy-4-[7-3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicacid, or[³H]-[1S-(1α,2β,3β,4α)]-4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicacid, or a salt or solvate of any one thereof.

Advantageously, the radioligand has a specific activity of greater than10 Ci/mmol and a P2Y_(ADP) receptor activity (IC₅₀) of less than 1micromolar (μM).

In the context of the present specification, “specific activity” isdefined as the activity per unit mass of substance containing aradioactive nuclide and is normally expressed as millicuries permilligram (mCi/mg) (kBq/mg), as millicuries per millimole (mCi/mmol)(kBq/mmol), or as curies per millimole (Ci/mmol) (GBq/mmol); and“P2Y_(ADP) receptor activity (IC₅₀)” is defined as the concentration,expressed in micromolar units, of radioligand required to inhibit themaximal aggregation response elicited by ADP according to the plateletaggregation assay as described in WO 98/28300. The platelet aggregationassay, which uses washed human platelets, is carried out in thefollowing manner.

Human venous blood (100 ml) is divided equally between 3 tubes, eachcontaining 3.2% trisodium citrate (4 ml) as anticoagulant. The tubes arecentrifuged for 15 minutes at 240 G to obtain a platelet-rich plasma(PRP) to which 300 ng/ml prostacyclin is added to stabilize theplatelets during the washing procedure. Red cell free PRP is obtained bycentrifugation for 10 minutes at 125 G followed by furthercentrifugation for 15 minutes at 640 G. The supernatant is discarded andthe platelet pellet resuspended in modified, Calcium Free Tyrodesolution (10 ml) (CFT), composition: NaCl 137 mM, NaHCO₃ 11.9 mM,NaH₂PO₄ 0.4 mM, KCl 2.7 mM, MgCl₂ 1.1 mM, dextrose 5.6 mM, gassed with95% O₂/5% CO₂ and maintained at 37° C. Following addition of a further300 ng/ml PGI₂, the pooled. suspension is centrifuged once more for 15minutes at 640 G. The supernatant is discarded and the platelets areresuspended initially in 10 ml CFT with further CFT added to adjust thefinal platelet count to 2×10⁵/ml. This final suspension is stored in a60 ml syringe at 3° C. with air excluded. To allow recovery fromPGI₂-inhibition of normal function, platelets are used in aggregationstudies no sooner than 2 hours after final resuspension.

Aliquots of platelet suspension (3 ml) are added to tubes containingCaCl₂ solution (60 μl of 50 mM solution with a final concentration of 1mM). Human fibrinogen (Sigma, F 4883) and 8-sulphophenyltheophylline(8-SPT, which is used to block any P₁-agonist activity of testsubstance) are added to give final concentrations of 0.2 mg/ml (60 μl of10 mg/ml solution of clottable protein in saline) and 300 nM (10 μl of15 mM solution in 6% glucose), respectively. Platelets or buffer asappropriate are added in a volume of 150 μl to the individual wells of a96 well plate. All measurements are made in triplicate in platelets fromeach donor.

Aggregation responses in 96 well plates are measured using the change inabsorbance given by the plate reader at 660 nm. Either a Bio-Tec Ceres900C or a Dynatech MRX are used as the plate reader.

The absorbance of each well in the plate is read at 660 nm to establisha baseline figure. Saline or the appropriate solution of test substance(e.g. the radioligand) is added to each well in a volume of 10 gl togive a final concentration of 0, 0.01, 0.1, 1, 10 or 100 mM. The plateis then shaken for 5 minutes on an orbital shaker on setting 10 and theabsorbance read at 660 nm. Aggregation at this point is indicative ofagonist activity of the test substance. Saline or ADP (30 mM; 10 μl of450 mM) is then added to each well and the plate shaken for a further 5minutes before reading the absorbance again at 660 nm. The concentrationof test substance that produces a response which is half the maximumcontrol ADP response is the IC₅₀ value.

According to a preferred embodiment of the invention, the competitionbinding assay comprises

(i) isolating and washing human platelets or human platelet membranes,

(ii) incubating the platelets or platelet membranes with a P2Y_(ADP)receptor radioligand and a candidate P2Y_(ADP) receptor ligand,

(iii) filtering and washing the platelets or platelet membranes, and

(iv) measuring bound radioactivity.

In step (i) above, methods for isolating and washing human platelets orhuman platelet membranes are known in the art, e.g. as described byConnolly et al. (1992), J. Biol. Chem., 267, 6893-6898 and Biochim. etBiophys. Act. (1986), 854, 67-76.

In step (ii), the incubation is conveniently carried at a temperature inthe range from 4 to 37° C., for a period of time of from 5 to 120minutes.

The present invention further provides the use of[¹²⁵I]-[1S-[1α,2β,3β,4α(E)]]-2,3-dihydroxy-4-[7-(3-iodo-prop-2-enylarnino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicacid, or[³H]-[1S-(1α,2β,3β,4α)]-4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicacid, or a salt or solvate of any one thereof, as a P2Y_(ADP) receptorradioligand in a competition binding assay as hereinbefore defined.

The present invention will now be further illustrated by reference tothe following Examples.

EXAMPLE 1 Synthesis of[³H]-[1S-(1α,2β,3β,4α]-4-[7-(Butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrinidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicAcid, Sodium Salt

A flask containing[1R-[1α(E),2β,3β,4α]]-3-[4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-2,3-dihydroxycyclopentyl]-2-propenoicacid (5.5 mg) (prepared as described in Example 3 of WO 97/03084) inethanol (1 ml), containing palladium on carbon (5% w/w Pd, 1 mg) wasattached to a tritium manifold, evacuated and tritium gas (10 Ci, 57.6Ci mmol⁻¹, 174 μmol) introduced. The reaction was stirred for 18 hoursat room temperature, then the catalyst removed by filtration and theremaining tritium removed by lyophilisation with ethanol (2×1 ml).Purification (HPLC, Symmetry C8, 45% acetonitrile/0.025% v/v aqueousacetic acid as eluant) gave the title acid which was converted to thesodium salt. The salt was dissolved in ethanol (16ml) to afford asolution of the title compound (1 mCi ml⁻¹, 24 Ci mmol⁻¹). The productwas characterised by comparison with unlabelled compound (prepared asdescribed in Example 3 of WO 97/03084) (HPLC, Symmetry C8, 50%methanol/0.1% w/v aqueous ammonium acetate to 95% methanol over 10minutes as eluant).

EXAMPLE 2 Synthesis of [¹²⁵I]-[1S-[1α,2β,3β,4α(E)]]-2,3-Dihydroxy-4-[7-(3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicAcid

[3aR-[3aα,4α,6α(E),6aα]]-6-[7-(3-Tributylstannyl-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-tetrahydro-2,2-dimethyl-4H-cyclopenta-1,3-dioxole-4-carboxylicacid (1.66 nmol) (prepared as described in Example 93 of WO 98/28300) inacetonitrile (40 μl) was added to sodium [¹²⁵I Iiodide (obtained fromAmersham International) (1 mCi, 10 μl, 0.5 nmol), followed by a solutionof chloramine-T in water (7 μl, 1.5 nmol). The vial was sealed, shakenvigorously, then left to stand at room temperature for 1 hour. Aqueoustrifluoroacetic acid (25% v/v, 50 μl) was then added and the reactionmixture resealed, shaken and left to stand for a further 2 hours.Purification (HPLC, Novapak C18, 30% acetonitrile/0.5% w/v aqueousammonium actate then increased to 95% acetonitrile as eluant) gave thetitle compound, to which ethanol was added to the required volume. Theproduct was characterised by comparison with unlabelled compound(prepared as described in Example 93 of WO 98/28300) (HPLC symmetryshield C8 75×3.9 mm, 25% acetonitrile/0.5% w/v aqueous ammonium acetateincreased to 95% acetonitrile over 3 minutes and held for 3 minutes, 2ml min⁻¹, retention time=2.98 minutes).

EXAMPLE 3 Washed Platelet Prearation

Human venous blood (100 ml), obtained from healthy volunteers wasdivided equally between 3 tubes, each containing 3.2% trisodium citrate(4 ml) as an anti-coagulant. The tubes were centrifuged for 15 min at240 G to obtain platelet-rich plasma (PRP) to which prostacyclin (PGI₂300 ng·ml⁻¹) was added to stabilize platelets during the washingprocedure. Red cell-free PRP was obtained by centrifugation for 10minutes at 125 G and following further centrifugation for 15 minutes at640 G, the supernatant was discarded and the platelet pellet in eachtube resuspended in modified, calcium-free, Tyrodes solution (10 ml)(CFT, composition: NaCl, 137.0 mM; NaHCO₃, 11.9 mM; NaH₂PO₄, 0.4 mM;KCl, 2.7 mM; MgCl₂, 1.1 mM and dextrose, 5.55 mM), gassed with 95% O₂/5%CO₂ and maintained at 37° C. Following addition of PGI₂ (300 ng·ml⁻¹),the pooled suspension was centrifuged once more for 15 minutes at 640 G.The supernatant was discarded and the platelets resuspended in CFT togive a final platelet count of 200-250×10³.μl⁻¹. The platelets were usedwithin 30 min for radioligand binding studies.

EXAMPLE 4 Binding Assays

Binding assays were performed in 96-well plates, with each wellcontaining 250 μl aliquots of CFT consisting of 50 μl 0.1μCi[¹²⁵I]-[1S-[(1α,2β,3β,4α(E)]]-2,3-dihydroxy4-[7-(3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicacid (final concentration 0.18 nM) or 50 μl 0.1μCi[³H][1S-(1α,2β,3β,4α)]-4-[7-(Butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicacid, sodium salt (final concentration 17 nM) and 200 μl of washedplatelets at a concentration of 200-250×10³.μl⁻¹ (final concentration160-200×10³.μl⁻¹). All putative P2Y_(ADP) ligands were tested induplicate over the appropriate concentration range by addition of 5 μlof compound prior to adding the radioligand, with appropriate solventcontrols being performed in parallel. The plates were incubated for 30min at room temperature on a plate shaker (Stuart scientific; model S01,setting 6) prior to terminating the reactions by filtration. Filtrationwas performed using a MACHIII cell harvester with 2×2s wash periods(with CFT) on to Whatman GF/B filter plates for platelets incubated[¹²⁵I[1S-[1α,2β,3β,4α(E)]]-2,3-dihydroxy-4-[7-(3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicacid or a Wallac cell harvester using glass fibre printed filtermatstype A, with a 7s wash time (with CFT) for platelets incubated with[³H]-[1S-(1α,2β,3β,4α) ]-4-[7-(Butylamino)-5-(propylthio)-3H-

1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoic acid, sodiumsalt. The resultant filterplates were then, in the case of the MACHI,sealed and Microscint 20 (50 gl) added prior to determination of [¹²⁵I]levels by scintillation counting on a Packard Topcounter or, in the caseof the Wallac filtermats, the individual wells were punched into vialscontaining 5 ml scintillation fluid for determination of [³H] levels ina beta counter.

Non-specific binding was determined in the presence of the standardP2Y_(ADP) antagonist, 2-propylthio-D-β,γ-dichloromethylene ATP (10 μM),as described by Humphries et al., Br. J. Pharmacology (1995), 115,1110-1116.

Results were expressed as specific binding in CPM/DPM and werecalculated by subtracting the non-specific binding from the totalbinding achieved at each concentration. For each test compound, abinding affinity (IC₅₀) was calculated by linear interpolation of theconcentration/inhibition curve, using the software package Excel. TheIC₅₀ value being the concentration at which a 50% reduction in specificbinding of either[¹²⁵I[-]1S-[1α,2β,3β,4α(E)]]-2,3-dihydroxy-4-[7-(3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicacid or[³H]-[1S-(1α,2β,3β,4α)]-4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrirnidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicacid, sodium salt was achieved. Results were reported as pKi valueswhich are equal to the negative, logarithm of the IC₅₀ (pIC₅₀) in thissystem (Cheng Prusoff). In cases where there was insufficientdisplacement of binding to calculate a pKi value, the activities werereported as being <6.

What is claimed is:
 1. A competition binding assay to identify aP2Y_(ADP) receptor ligand, which comprises contacting a P2Y_(ADP)receptor with a P2Y_(ADP) receptor radioligand and a candidate P2Y_(ADP)receptor ligand, and measuring reactivity bound to the P2Y_(ADP)receptor, wherein the radioligand is selected from the group consistingof [¹²⁵I]-1S-[1a,2b,3b,4a(E)]]-2,3-dihydroxy-4-[7-(3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicacid,[³H]-[1S-(1α,2β,3β,4α)]-4-[7(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicacid, salts and solvates thereof.
 2. An assay according to claim 1,wherein the P2Y_(ADP) receptor is of human origin.
 3. An assay accordingto claim 2 which comprises: (i) isolating and washing human platelets orhuman platelet membranes, (ii) incubating the platelets or plateletmembranes with a P2Y_(ADP) receptor radioligand and a candidateP2Y_(ADP) receptor ligand, (iii) filtering and washing the platelets orplatelet membranes, and (iv) measuring radioactivity bound to theplatelets or platelet membranes.
 4. An assay according to claim 1,wherein the radioligand has a specific activity greater than 10 Ci/mmoland a P2Y_(ADP) receptor activity (IC₅₀) of less than 1 micromolar (μM).5. [¹²⁵I]-1S-[1α,2β,3β,4α(E)]]2,3-dihydroxy-4-[7-(3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin3-yl]-cyclopentanecarboxylicacid, salts and solvates thereof.
 6. [³H]-[1S-(1α,2β,3β,4α)]-4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicacid, salts and solvates thereof. 7.[¹²⁵I]-1S-[1α,2β,3β,4α(E)]]2,3-dihydroxy-4-[7-(3-iodo-prop-2-enylamino)-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-cyclopentanecarboxylicacid, salts and solvates thereof, having a specific activity greaterthan 10 Ci/mmol. 8.[³H]-[1S-(1α,2β,3β,4α)]4-[7-(butylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]pyrimidin-3-yl]-2,3-dihydroxy-cyclopentanepropanoicacid, salts and solvates thereof, having a specific activity greaterthan 10 Ci/mmol.